1. Prior Art
Generally, enzyme proteins are water soluble and enzyme reactions are usually carried out in the solubilized state. Enzyme reactions cannot be done continuously on an industrial scale due to above mentioned properties of enzymes. Also, separation and purification of the reaction product cannot be conducted effectively.
Therefore, to resolve these problems many attempts have been made for insolubilizing enzymes. Methods which have been developed so far can be divided into four groups, i.e. adsorption method, carrier binding method, crosslinking method and entrapping method.
However, these prior art methods have disadvantages:
(1) Adsorption or binding of enzyme to carrier is not always efficient. Consequently, the enzyme leaks frequently. (2) Enzyme activity is low and unstable. (3) Manufacturing process is tedious. (4) They can be applied only to a highly purified enzyme.
When the radiopolymerization of water soluble monomers such as acrylamide is conducted, oxygen was known to inhibit the radial polymerization, therefore until now when radiopolymerization is carried out the solution to be polymerized is usually bubbled with Nitrogen or Argon. Thus, from the solution and its surroundings, oxygen is completely expelled, and then finally radio-polymerization is conducted.
According to the above-mentioned prior art method, namely under anaerobic and unfrozen conditions, radio-polymerization was conducted. According to the present invention, however, it is necessary to expel oxygen from the solution and its surroundings, thus making the process very simple and having a great advantage over the prior art process.
Because of the difference in the new method of the present invention and the old method of the prior art, the final product obtained also is very different. When radio polymerization is conducted without freezing (as in the prior art), the polymer is obtained as a curdy precipitate, but according to the method of the present invention the solution is polymerized as a whole, like spongy mass. This gives a product of more uniform consistency.
The very substantial difference between the present invention and the process disclosed in the prior art has been explained hereinabove. Further, according to the present invention, radio polymerization as shown in the examples of the specification was conducted under aerobic conditions at the frozen state and thus this process of the present invention is very useful.
2. Summary of the Invention
The present invention relates to a method for producing water-insoluble enzymes so as to obtain water-insoluble enzymes having high activity and stability by a simple method. That is to say, our invention comprises freezing a solution of one or more monomers and enzyme, and then polymerizing the resultant composition by irradiating same with a dose of about 40 - 100 Krad of gamma ray. This method entraps the active enzyme within the resulted polymer lattice. Before freezing the solution an organic substance can be incorporated which promotes the polymerization yield and protects the highly purified enzyme from radiation inactivation. Further, our invention includes a new improved technique of drying under vacuum (lyophilization) immediately after irradiating the frozen solution.